1. Field of the Invention
The present invention is directed to a purified, biologically-derived carboxylic acid reductase, and to methods of using the carboxylic acid reductase as a biocatalyst for the reduction of carboxylic acids or their derivatives to their corresponding useful product(s).
2. Description of Related Art
Microorganism-produced enzymes are widely used as a class of biocatalytic reagents in synthetic organic chemistry in a wide variety of reactions including, e.g., oxidations, reductions, hydrolyses, and carbon--carbon bond ligations. Enzyme reactions, such as those catalyzed by esterases, for example, may be used either hydrolytically or to synthesize esters, depending on whether the reaction medium is aqueous or organic in composition.
Biocatalysts are valued for their intrinsic abilities to bind organic substrates and to catalyze highly specific and selective reactions under the mildest of reaction conditions. These selectivities and specificities are realized because of highly rigid interactions occurring between the enzyme active site and the substrate molecule. Biocatalytic reactions are particularly useful when they may be used to overcome difficulties encountered in catalysis achieved by the use of traditional chemical approaches.
The reduction of carboxylic acids by microorganisms is a relatively new biocatalytic reaction that has not yet been widely examined or exploited. Jezo and Zemek reported the reduction of aromatic acids to their corresponding benzaldehyde derivatives by Actinomycetes in Chem. Papers 40(2):279-281 (1986). Kato et al. reported the reduction of benzoate to benzyl alcohol by Nocardia asteroides JCM 3016 (Agric. Biol. Chem. 52(7):1885-1886 (1988)), and Tsuda et al. described the reduction of 2-aryloxyacetic acids (Agric. Biol. Chem. 48(5): 1373-1374 (1984)) and arylpropionates (Chem. Pharm. Bull. 33(11):4657-4661 (1985)) by species of Glomerella and Gloeosporium. Microbial reductions of aromatic carboxylic acids, typically to their corresponding alcohols, have also been observed with whole cell biotransformations by Clostridium thermoaceticum (White et al., Eur. J. Biochem. 184:89-96 (1989)), and by Neurospora (Bachman et al., Arch. Biochem. Biophys. 91:326 (1960)). More recently, carboxylic acid reduction reactions have reportedly been catalyzed by whole cell preparations of Aspergillus niger, Corynespora melonis and Coriolus (Arfmann et al., Z. Naturforsch 48c:52-57 (1993); cf., Raman et al., J. Bacterial 84:1340-1341 (1962)), and by Nocardia (Chen and Rosazza, Appl. Environ. Microbiol. 60(4):1292-1296 (1994)).
Carboxylic acid reductases are complex, multicomponent enzyme systems, requiring the initial activation of carboxylic acids via formation of AMP and often coenzyme A intermediates (see, e.g., Hempel et al., Protein Sci. 2:1890-1900 (1993). However, an enzymatic reaction offers significant advantages over existing methods used in chemical reductions of carboxylic acids, or their derivatives. Unlike many substrates subjected to biocatalytic reactions, carboxylic acids are generally water soluble, rendering them of potentially broad application to this class of enzyme. The carboxylic acid reduction reaction appears to bear the usual desirable features of functional group specificity. It also functions well under mild reaction conditions and produces a high yield of product. The reduction of the activated carboxylic acid intermediate occurs step-wise to give aldehyde, and then alcohol products (Gross et al., Eur. J. Biochem. 8:413-419; 420-425 (1969); Gross, Eur. J. Biochem. 31:585-592 (1972)).
In the present inventor's laboratory, whole cell preparations of Nocardia sp. NRRL 5646 were found to be highly enantioselective in the reduction of isomeric ibuprofen substrates (Appl. Environ. Microbiol. 60(4):1292 (1994)). However, with this organism, the substrate specificity for carboxylic acids was significantly different than that which had been reported by Kato et al. for Nocardia asteroides in Agric. Biol. Chem. 52(7):1885 (1988), and by others. Consequently, recognizing the importance of identifying and understanding the biocatalytic enzyme capable of reducing a carboxylic acid to its aldehyde product, the inventors developed a method of rapid purification and characterization of the enzyme, and of determining its enantioselectivity and effect on a series of aryl-carboxylic acid substrates. The purified enzyme of the present invention was classified as an aryl-aldehyde oxidoreductase, also correctly denominated a carboxylic acid reductase (EC 1.2.1.30).
It will be appreciated that the availability of large quantities of the novel reductase made possible by the present invention, permits an crucial examination of the structure of the enzyme and an understanding of the mechanisms involved in the catalysis. In the case of many aldehydes, there has been a long-felt need to locate commercially viable methods for their production from plentiful, low-cost starting materials.
Vanillin is a classic example of such a product. Natural vanilla, one of the most important flavors used in the food industry, is presently extracted from the cured pods of the flowers of Vanilla planifolia. Yet because of the escalating cost of producing natural vanilla, methods are constantly being sought to manufacture vanillin (3-methoxy-4-hydroxybenzaldehyde), the most important organoleptic component in vanilla. Over 12,000 tons of vanillin are currently produced annually from byproducts of the petrochemical and wood pulping industries (Prince et al., Trends. Biol. Sci. 19:521(1994); Hagedorn et al., Ann. Rev. Microbiol. 48:773-800 (1994)). Nevertheless, the demand for natural and environmentally friendly products have spawned efforts to produce vanillin biochemically by microbial transformation from natural substrates including phenolic stibenes (Japanese Patent No. 2,195,871), eugenol (U.S. Pat. No. 5,017,388; Japanese Patent No. 5,227,980), and ferulic acid and benzenoid precursors (U.S. Pat. Nos. 5,262,315 and 5,128,253). However, despite the continuing effort to develop microbial transformations for vanillin production, the yields provided by the published methods are low, and the time for transformation is long.
In response to this need, the present invention provides not only a greater understanding of the metabolic pathways, cofactors and the enzymes needed to improve the yield of such valuable aldehyde and alcohol products by manipulating the metabolic network, but it also discloses for the first time, a safe and efficient method of producing GRAS vanillin from an inexpensive and abundant source, vanillic acid and its precursors, by a microbiologically-derived, purified, aryl-aldehyde oxidoreductase (carboxylic acid reductase). Moreover, further advancement of the art will be greatly enhanced by the inventors' purification of the enzyme, and the present invention will provide many new ways to study the mechanisms involved in biocatalysis.